This application focuses on the protozoan parasite Trypanosoma brucei, which causes devastating diseases in humans and animals in sub-Saharan Africa. There are no vaccines, and therapeutic drugs have serious side effects and decreasing efficacy. Thus, there is a pressing need for research to better understand the biology of these human pathogens and the mechanisms they use to survive within their hosts. T. brucei undergoes a complex life cycle between the mammalian host and the blood-feeding tsetse fly vector, which among others involves changes in cell morphology, surface coat composition, metabolism, signaling pathways and gene expression. Consequently, these parasites have evolved adaptations to allow for their survival in both the gut and salivary glands of the tsetse fly, as well as in the bloodstream of their mammalian host. By overexpressing a single RNA-binding protein (RBP6) in non- infectious trypanosomes, we recapitulated in vitro the events leading to acquisition of infectivity in the insect vector, including the expression of metacyclic variant surface glycoproteins (mVSGs). At present, little is known how mVSG gene expression is activated and how the expression is switched to bloodstream-form VSGs, once the parasite enters a mammalian host. One major goal of this application will be to examine how trypanosomes receive instructions to begin synthesizing the mVSG coat, how each cell expresses a single mVSG, and how mVSG expression is repressed and switched to the expression of bloodstream-form VSGs. We will apply a number of high-throughput approaches to monitor the chromatin structure of mVSG genes during developmental progression and test whether a second RNA-binding protein (RBP10) is a facilitator of [m]VSG expression. Primary transcripts and mature mRNAs will be monitored using RNA-Seq, the position, amount, and orientation of transcriptionally engaged RNA polymerase I will be surveyed by global run-on-sequencing (GRO-Seq) and transcription factor binding will be gauged by chromatin immunoprecipitation (ChIP) coupled with Illumina sequencing (ChIP-Seq). A second emphasis will be on RNA interference (RNAi). Since our discovery of RNAi in T. brucei in 1998, this pathway has been a focus of our investigations, which have led to the finding that RNAi functions both in the nucleus and in the cytoplasm and to the identification of five core RNAi genes. We will employ different approaches to address the question what defines the RNA-induced silencing complex (RISC), i.e. what other cellular factors functionally interact with the RNAi machinery, and how the levels of Argonaute are regulated. Finally, we will further address the biological function of RNAi by determining the RNAi targets during the T. brucei developmental cycle, which is now possible with our newly developed differentiation system.